Stefania Bonazzi

Acclimation of chloroplast transglutaminase to high NaClconcentration in a polyamine-deficient variant strain of Dunaliella salina and in its wild type

Summary The wild type (Wt) and the polyamine-deficient strain (PA − vs) of the halotolerant Dunaliella salina were subjected to stress caused by 3.5 mol/L NaCl concentration. The chloroplasts were isolated and the molecular aspects of their reaction to salt stress were studied together with their recovery response to these hyper-saline conditions. In the Wt, the photosynthetic complexes were found to be severely affected by salt stress under light conditions. Transglutaminases, which are present in chloroplasts as two units of 25 and 50 kDa, were immunorecognized by antibodies raised against rat prostatic gland transglutaminase. The amount, in particular that of the 50 kDa unit, underwent an immediate change following hyper-saline stress. These concentration changes were found to coincide with variations in enzymic activity, which is also affected by the presence or absence of light. The PA − vs has a concentration of proteins and chlorophylls which is much lower than that of the Wt. In addition, the PA − vs appeared to be more severely affected by both salt and subculture stresses. Its recovery time was also longer. Its TGase activity increased after salt stress and was always higher in the light than in the dark, except soon after subculture, showing an additive stress effect of salt and light. In the PA − vs acclimated to high salinity, or immediately after stress application, the chloroplast content of chlorophyll a and b was considerably enhanced, like the TGase activity (by two-fold or more), and these changes exhibited almost coincident behaviours. Some transglutaminase substrates (proteins of 68, 55, 29 and 27 kDa) were found to be similar to those present in higher plants (thylakoid photosynthetic complexes and Rubisco). They were more markedly labelled by [1,4- 14 C] polyamines when the transglutaminase assay was performed in the light than in the dark, and much more in algae already acclimated to hyper-saline conditions than in those cultured in the optimal saline medium, or subjected to stress. The amount of 68 and 55 kDa polypeptides was particularly high in the 3.5 mol/L NaCl acclimated cells. The possible role of polyamine conjugation in the assembly of chloroplast proteins in cells affected by salt stress is discussed.

Chromonic lyomesophases formed by the self-assembly of the cyclic dinucleotide d(cGpGp)

Abstract The cyclic dinucleotide d(cGpGp) undergoes a self-association process in water to give, first, columnar aggregates similar to the four-stranded helix of poly(G). Successively, at higher concentration, these aggregates self-organize to give a cholesteric and a hexagonal mesophase, the former of which appears only in biphasic systems. The self-assembly process in isotropic solution has been studied by CD spectroscopy and the structure of the mesophases was investigated by optical microscopy and X-ray diffraction.

Recovery of growth capacity and of chloroplast transglutaminase activity induced by polyamines in a polyamine-deficient variant strain of Dunaliella salina

Summary The variant strain of Dunaliella salina is characterised by: a) a very low growth capacity; b) a deficiency in polyamines, transglutaminase chlorophyll, and protein content; and c) an unbalanced putrescine/spermidine ratio. A supply of 100 μmol/L putrescine or 1 mmol/L ornithine induces the resumption of growth. Putrescine has several additional effects: endogenous putrescine and spermidine levels increase and their ratio reaches 1.6, a value typical of the growing wild type; chlorophyll a and b also increase and their ratio (3.6) becomes close to that of the wild type (3.0); protein content increases, especially in the chloroplast. Transglutaminase(s) in chloroplasts of putrescine-treated algae is stimulated and increases its conjugating activity by 9-fold in the light and less than 3-fold in the dark when compared to untreated controls. The role of polyamines in improving the physiological condition of the chloroplast and their effect on growth are discussed.

The self‐assembly and liquid crystal formation of d(GpGpApGpG)

d(GpGpApGpG) dissolved in water forms liquid crystalline phases of the cholesteric and hexagonal type. The building blocks of the phases are columnar four‐stranded aggregates composed of G quartets. The diameter of the columnar aggregates is larger and the melting temperature is lower than for homoguanylic derivatives, indicating a distorsion and loss of stability due to the presence of adenines, which do not form a hydrogen‐bonded quartet. The present study shows that, as often observed for single crystals, it is possible also in the liquid crystalline phase to construct long columnar aggregates composed of shorter segments of the blunt‐end type in a “closed architecture.”

A study of the self-assembly of 2-deoxyguanosine 3 5-cyclic monophosphate, d(cGp), by CD and X-ray diffraction

2-Deoxyguanosine 3 5-cyclic monophosphate forms in water cholesteric and hexagonal columnar mesophases. The polymorphic behaviour and the structural building blocks of the liquid crystalline phases, as determined by optical microscopy, CD spectroscopy and X-ray diffraction, are comparable to those found with all the deoxyguanylates investigated so far (in particular with deoxuguanosine 5- and 3-monophosphate). The present results show that the formation of a stacked array of planar G-tetramers, a necessary condition for the existence of the columnar mesophases, occurs even in the absence of hydrogen bonding groups linking the molecules along the length of the columns.